1. Field of the Invention
The present invention relates to chemotaxis chambers, i.e., to chambers for measuring the effect of concentration gradients of diffusible chemicals upon the directional response of biological cells. More specifically, the present invention relates to chemotaxis chambers comprising two compartments separated by a thin passage, used to study the migration or orientation of adherent mobile cells.
2. Background of the Invention
Chemotaxis is the directional response of biological cells or organisms to concentration gradients of mobile chemicals. Conventional chemotaxis chambers comprise two compartments separated by a filter, with one or both of the compartments open to air. Cells in suspension are placed in the upper compartment, and a chemotactic factor or control is placed in the bottom compartment. The chemotactic factor can be used in various dilutions to get a dose-response curve. The controls are generally of two kinds: negative, when the same medium is used to suspend the cells above and below the filter; and chemokinetic, when a chemotactic factor is placed in the same concentration in the medium with the cells and on the opposite side of the filter. Chemokinetic controls allow the user to distinguish heightened random activity of the cells, due to contact with the chemotactic factor, from directional response in a concentration gradient of the chemotactic factor.
Chemotactic activity is measured by first establishing a stable concentration gradient in the chemotaxis chamber. The chamber is incubated for a predetermined time, then the filter is removed from the apparatus. The cells that have migrated through the filter (or into the filter to a certain depth) are then counted. A comparison is then made between the activity of the cells in a concentration gradient of the chemotactic factor being tested, and the activity of the cells in the absence of the concentration gradient.
The apparatus can also be used to measure the response of cells of different origins--e.g., immune cells from patients suffering from diseases--to a chemotactic factor of known chemotactic activity. In this case the cells in question are challenged by both a negative control and the chemotactic factor to see if the differential response is depressed or normal.
Cell orientation in response to a concentration gradient of chemotactic factor can be determined using a Zigmond chamber. A Zigmond chamber is a 2 mm thick microscope slide with two 1 mm deep.times.4 mm wide grooves ground across the slide, leaving a 1 mm wide unground section between the grooves. Cells are allowed to adhere to a microscope coverglass, which is then inverted so that the cells and some fluid media are positioned over the 1 mm wide unground section of the slide. The coverglass is clamped in this position at each end of the slide, chemotactic factor is pipetted into one of the two grooves, and media is pipetted into the other groove. Capillary action draws the fluid into the grooves. The slide is incubated at 37.+-.1.degree.0C. for about 30 minutes. It is then viewed under a microscope at a magnification of 100 to 1,000 X, and the extent of cell orientation is observed. Migration of the cells can also be studied using elaborate time-lapse image analyzers. Usually, however, the chamber is only used to check the proportion of cells that orient towards a chemotactic factor concentration gradient, compared to the proportion of oriented cells in a control chamber that does not have a chemotactic factor concentration gradient.
Microchemotaxis chambers and some of their applications are described in Falk et al., 37 A 48 Well Micro Chemotaxis Assembly for Rapid and Accurate Measurement of Leukocyte Migration," Journal of Immunological Methods, 33, 239-247 (1980); Harvath et al., "Rapid Quantification of Neutrophil Chemotaxis: Use of a Polyvinylpyrrolidone-free Polycarbonate Membrane in a Multiwell Assembly," Journal of Immunological Methods, 37, 39-45 (1980); Richards et al., 37 A Modified Microchamber Method for Chemotaxis and Chemokinesis," Immunological Communications, 13 (1), 49-62 (1984); Falk et al., "Only the Chemotactic Subpopulation of Human Blood Monocutes Expresses Receptors for the Chemotactic Peptide N-Formylmethionyl-Leucyl-Phenylalanine," Infection and Immunity, 36, 450-454 (1982); Zigmond, Sally, Gatewood, Beth and Joe, John, "CD45 is Not Involved in the Processing of Spatial Information Required for Chemotaxis," Journal of Immunology (to be published); Zigmond, Sally and Lauffenburger, Douglas A., "Assays of Leukocyte Chemotaxis," Annual Reviews of Medicine, 37, 149-155 and Harvath et al., "Two Neutrophil Populations in Human Blood with Different Chemotactic Activities: Separation and Chemoattractant Binding," Infection and Immunity, 36 (2), 443-449 (1982), all of which are expressly incorporated by reference herein.
Multiple-site chemotaxis chambers are described in patent application Ser. No. 07/672,561, filed on Mar. 20, 1991, which is expressly incorporated by reference herein.